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Properties of NADH oxidase from Lactobacillus delbrueckii ssp bulgaricus
Author(s) -
Yi Xianwen,
Kot Eva,
Bezkorovainy Anatoly
Publication year - 1998
Publication title -
journal of the science of food and agriculture
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.782
H-Index - 142
eISSN - 1097-0010
pISSN - 0022-5142
DOI - 10.1002/(sici)1097-0010(199812)78:4<527::aid-jsfa149>3.0.co;2-l
Subject(s) - biochemistry , size exclusion chromatography , chemistry , tris , nad+ kinase , enzyme , chromatography , hydrogen peroxide , lactobacillus , agarose , polyacrylamide gel electrophoresis , fermentation
Lactobacillus delbrueckii spp bulgaricus produces substantial amounts of hydrogen peroxide, yet the enzymology of this process has not been explored. We have partially purified a NADH oxidase (EC 1.6.99.3) from the soluble cell fraction (cytosol) of L delbrueckii ssp bulgaricus , prepared by sonication of the microorganisms in a 0·1 M Tris buffer at pH 7·2. The cytosol was treated with agarose beads containing attached FAD residues, which served to bind the enzyme. The enzyme was eluted with a 0·01 M Tris buffer at pH 7·2 containing 10 μ M FAD. This preparation had 0·2 mg ml −1 protein and showed a single major band in a polyacrylamide gel electrophoresis system with a molecular weight of near 30 kDa. Gel filtration chromatography gave a molecular weight of 25 kDa. This enzyme preparation consisted of 4–6 subunits with a molecular weight of 5–6 kDa each, which were held together by FAD. No EDTA‐sensitive component was required for activity. The enzyme used β‐NADH, but not α‐NADH or β‐NADPH, to produce stoichiometric amounts of H 2 O 2 . The apparent K m with respect to NADH, using production of NAD as a criterion, was 534 μ M . The enzyme was active at 4°C and was quite thermostable. Its pH optimum was around pH 7·2. The metabolic function of NADH oxidase in lactobacilli remains obscure, though the H 2 O 2 it generates may have a profound effect on the viability of other organisms. © 1998 Society of Chemical Industry.